Triazole derivatives useful in therapy

ABSTRACT

The invention provides compounds of formula I, 
     
       
         R 1 —OP(O)(OH) 2   I 
       
     
     wherein R 1  represents the non-hydroxy portion of a triazole antifungal compound of the type comprising a tertiary hydroxy group; or a pharmaceutically acceptable salt thereof. 
     The compounds of the invention are useful in the treatment of fungal infections, and have good aqueous solubility.

This application is a continuation of a non-provisional application Ser.No. 09/117,175, filed 8 Jan. 1999which is the National Stage ofInternational Application No. PCT/EP97/00445, filed 27 Jan. 1997 nowabandoned, which is published in English under International publicationNo. WO 97/28169, and which claims priority from Great Britainapplication No. 9602080.5, filed 2 Feb. 1996.

This invention relates to triazole derivatives useful in therapy (inparticular in the treatment of fungal infections in humans and othermammals), methods for their use, formulations including them andprocesses for their production.

A large number of triazole antifungal compounds are known. For example,European Patent Application 0440372, Example 7, discloses (2R,3S)-2-(2,4-difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1-(1H-1,2,4-triazol-1-yl)-butan-2-ol(also known as voriconazole) which has particularly good activityagainst the clinically important Aspergillus spp fungi. However, thecompound has low solubility in aqueous media, necessitating the use ofcomplexing agents to achieve satisfactory aqueous formulations, such asintravenous formulations. European Patent Application 0440372 suggestsco-formulation with cyclodextrin derivatives to improve solubility;however, it is always desirable to keep the number of ingredients in aformulation to a minimum so as to minimize possible adverse reactions inpatients.

UK Patent Application 2,128,193 discloses phosphoric acid esters for useas plant fungicides and insecticides.

Maurin et al [Int J Pharm, 1993, 94(1-3), 11-14] discloseα-(2,4-difluorophenyl)-α-[(1-(2-(3pyridyl)phenylethenyl)]-1H-1,2,4-triazole-1-ethanolbismesylate, which is stated to be an antifungal agent having highsolubility.

Other triazole antifungal agents are known from European PatentApplication 0576201 and international Patent Application WO 97/01552.

European Patent Application 0413674 discloses formation of prodrugs oftherapeutic glycosidase inhibitors by phophorylating a free hydroxygroup in the molecule. However, phosphorylation of tertiary hydroxygroups is not described.

It has now been found that triazole antifungal compounds of the typecomprising a tertiary hydroxy group, including(2R,3S)-2-(2,4-difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1-(1H-1,2,4-triazol-1-yl)-butan-2-ol,may be converted into pro-drugs having greatly enhanced solubility, butwhich are converted readily in vivo to give the desired active moiety.

According to the invention, there is provided a compound of formula I,

R¹—OP(O)(OH)₂  I

wherein R¹ represents the non-hydroxy portion of a triazole antifungalcompound of the type comprising a tertiary hydroxy group;

or a pharmaceutically acceptable salt thereof (referred to herein as“the compounds of the invention”).

The compounds of the invention are distinct from the prior art becausethe tertiary hydroxy group in triazole antifungal compounds of this typehas not previously lent itself to functionalization.

Pharmaceutically acceptable salts that may be mentioned include alkalimetal salts of the phosphate group, for example disodium or dipotassiumsalts; and salts with an amine counter ion, for example ethylenediamine,glycine or choline salts.

Preferably, R¹ represents a group of formula Ia,

in which

R² represents phenyl substituted by one or more halogen atoms;

R³ represents H or CH₃;

R^(3a) represents H, or together with R³ it may represent ═CH₂; and

R⁴ represents a 5- or 6-membered nitrogen-containing heterocyclic ringwhich is optionally substituted by one or more groups selected fromhalogen, ═O, phenyl [substituted by a group selected from CN and(C₆H₄)—OCH₂CF₂CHF₂] or CH=CH—(C₆H₄)—OCH₂CF₂CHF₂; or phenyl substitutedby one or more groups selected from halogen and methylpyrazolyl.

When R¹ represents a group of formula Ia, as defined above, R² ispreferably 2,4-difluorophenyl, and R³ is preferably H or methyl.

Nitrogen-containing heterocyclic rings that R⁴ may represent or compriseinclude triazolyl, pyrimidinyl and thiazolyl.

Preferred specific groups that R¹ may represent include:

The triazole antifungal compounds corresponding to the groups (a)-(g)above are: (a) D-0870 (under development by Zeneca, see also Example 19,European Patent Application 0472392); (b) fluconazole (sold by Pfizer,see also UK Patent Application 2099818); (c) Example 7 of EuropeanPatent Application 0440372, also known as voriconazole; (d) Example 35of U.S. Pat. No. 4,952,232; (e) the compound of Example 8 of the presentapplication; (f) Compound A of WO 95/22973 (see page 29), originallydisclosed as Compound 30 in Example 27 of EP 567982; and (g) ER-30346(see Drugs of the Future, 1996, 21(1): 20-24, Tetrahedron Letters, Vol37, 45, pp 8117-8120, 1996 AND European Patent Application 0667346,Example 88).

The present invention also provides a process for the production of acompound of formula I, as defined above, or a pharmaceuticallyacceptable salt thereof, which comprises phosphorylating a compound offormula II,

R¹OH  II

wherein R¹ is as defined above;

and where desired or necessary converting the resulting compound into apharmaceutically acceptable salt or vice versa.

The phosphorylation may be carried out using the following steps(1)-(3):

(1) Reacting a compound of formula II, as defined above, with a compoundof formula III,

R^(a)R^(b)N—P(OR^(c))(OR^(d))  III

wherein R^(a) and R^(b) independently represent C₁₋₆ alkyl, phenyl orsubstituted phenyl, or together with the nitrogen atom to which they areattached they may represent a ring such as a morpholine ring; and R^(c)and R^(d) independently represent hydroxy protecting groups selectedfrom benzyl optionally subsituted by one or more halogen atoms; to givea phosphite compound of formula IV,

R^(i)—O—P(OR^(c))(OR^(d))  IV

wherein R^(i), R^(c) and R^(d) are as defined above.

The reaction may be carried out in a solvent which does not adverselyaffect the reaction (e.g. methylene chloride) in the presence of a mildacid (for example tetrazole, 5-methyltetrazole or pyridiniumhydrobromide) and optionally 4-dimethylaminopyridine, at roomtemperature or above.

(2) Reacting the resulting phosphite of formula IV with an oxidant (forexample a peracid such as 3-chloroperoxybenzoic acid, or H₂O₂), to givea phosphate of formula V,

R¹—OP(O)(OR^(c))(OR^(d))  V

wherein R¹, R^(c) and R^(d) are as defined above. The reaction may becarried out in a solvent which does not adversely affect the reaction(e.g. methylene chloride or ethyl acetate) below room temperature (forexample 0-−20° C.).

(3) Removing the hydroxy protecting groups from the compound of formulaV to give a compound of formula I, as defined above.

As an alternative to step (1), phosphites of formula IV may be preparedaccording to steps (1A) and (1B):

(1A) Reaction of a compound of formula II, as defined above, with PCl₃in the presence of a base to give a postulated intermediate compound offormula VI,

R¹—O—PCl₂  VI

wherein R¹ is as defined above. The reaction may be carried out in asolvent which does not adversely affect the reaction (e.g. methylenechloride or ethyl acetate) at a temperature in the range −20 to +20° C.(for example 0° C.). Suitable bases include pyridine andN-methylimidazole.

(1B) Reaction of the compound of formula VI with a compound of formulaR^(c)OH and/or R^(d)OH (in which R^(c) and R^(d) are as defined above)to give a compound of formula IV, as defined above. The reaction isperformed without isolation of the compound of formula VI, at atemperature around room temperature.

Hydroxy protecting groups which R^(c) and R^(d) may represent include2,6-dichlorobenzyl and 2-chloro-6-fluorobenzyl. Benzyl groups may beremoved using catalytic hydrogenation (e.g. over Pearlman's catalyst orpalladium-on-carbon) or bromotrimethylsilane.

If step(3) is carried out in the presence of sodium acetate or sodiumhydroxide, the disodium salt may be obtained directly.

Process step (3) above, and the intermediate compounds of formula V fromfurther aspects of the invention. Compounds of formulae II and III areeither known or are available using known techniques.

It will be apparent to those skilled in the art that sensitivefunctional groups may need to be protected and deprotected duringsynthesis of a compound of the invention. This may be achieved byconventional techniques, for example as described in ‘Protective Groupsin Organic Synthesis’ by T W Greene and P G M Wuts, John Wiley and SonsInc, 1991.

The compounds of the invention are useful because they possesspharmacological activity in animals, including humans. In particular,the compounds are useful in the treatment or prevention of fungalinfections. For example, they are useful in treating topical fungalinfections in man caused by, among other organisms, species of Candida,Trichophyton, Microsporum or Epidermophyton, or in mucosal infectionscaused by Candida albicans (e.g. thrush and vaginal candidiasis). Theycan also be used in the treatment of systemic fungal infections causedby, for example, species of Candida (e.g. Candida albicans),Cryptococcus neoformans, Aspergillus flavus, Aspergillus fumigatus,Coccidioides, Paracoccidiodes, Histoplasma or Blastomyces.

Thus, according to another aspect of the invention, there is provided amethod of treatment or prevention of a fungal infection which comprisesadministering a therapeutically effective amount of a compound of theinvention to a patient. The use of the compounds of the invention aspharmaceuticals, and the use of the compounds of the invention in themanufacture of a medicament for the treatment or prevention of fungalinfections are also provided.

The in vitro evaluation of the antifungal activities of the compounds ofthe invention can be performed by determining the minimum inhibitoryconcentration (m.i.c.), which is the concentration of the testcompounds, in a suitable medium, at which growth of the particularmicro-organism fails to occur. In practice, a series of agar plates,each having the test compound incorporated at a particularconcentration, is inoculated with a standard culture of, for example,Candida albicans, and each plate is then incubated for 48 hours at 37°C. The plates are then examined for the presence or absence of growth ofthe fungus and the appropriate m.i.c. value is noted. Othermicro-organisms used in such tests can include Aspergillus fumigatus,Trichophyton spp., Microsporum spp., Epidermophyton floccosum,Coccidioides immitis and Torulopsis glabrata.

Some compounds of the invention, although active in vivo, may notdemonstrate activity in these in vitro tests.

The in vivo evaluation of the compounds of the invention can be carriedout at a series of dose levels by intraperitoneal or intravenousinjection, or by oral administration, to mice which are inoculated with,e.g. a strain of Candida albicans or Aspergillus fumigatus. Activity isbased on the survival of a treated group of mice after the death of anuntreated group of mice. The dose level at which the compound provides50% protection against the lethal effect of the infection (PD₅₀) isnoted. For Aspergillus spp. infection models, the number of mice curedof the infection after a set dose allows further assessment of activity.

For human use, the compounds of the invention can be administered alone,but will generally be administered in admixture with a pharmaceuticallyacceptable carrier selected with regard to the intended route ofadministration and standard pharmaceutical practice. For example, theycan be administered orally in the form of tablets containing suchexcipients as starch or lactose, or in capsules or ovules either aloneor in admixture with excipients, or in the form of elixirs, solutions orsuspensions containing flavouring or colouring agents. They can beinjected parenterally, for example, intravenously, intramuscularly orsubcutaneously. For parenteral administration, they are best used in theform of a sterile aqueous solution which may contain other substances,for example, enough salts or glucose to make the solution isotonic withblood.

For oral and parenteral administration to human patients, the dailydosage level of the compounds of the invention will be from 0.01 to 20mg/kg (in single or divided doses) when administered by either the oralor parenteral route. Thus tablets or capsules of the compounds willcontain from 5 mg to 0.5 g of active compound for administration singlyor two or more at a time, as appropriate. The physician in any eventwill determine the actual dosage which will be most suitable for anindividual patient and it will vary with the age, weight and response ofthe particular patient. The above dosages are exemplary of the averagecase; there can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

Alternatively, the compounds of the invention can be administered in theform of a suppository or pessary, or they may be applied topically inthe form of a lotion, solution, cream (for example comprising an aqueousemulsion of polyethylene glycols or liquid paraffin); or they can beincorporated, at a concentration between 1 and 10%, into an ointmentconsisting of a white wax or white soft paraffin base together with suchstabilizers and preservatives as may be required.

Thus, according to another aspect of the invention, there is provided apharmaceutical formulation including preferably less than 50% by weightof a compound of the invention in admixture with a pharmaceuticallyacceptable adjuvant, diluent or carrier. Aqueous intravenousformulations are of particular interest.

The invention is illustrated by the following Examples.

EXAMPLE 1

2-(2,4-Difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)-2-propylDihydrogen Phosphate

(a) Dibenzyl2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)-2-propyl Phosphate

A solution of2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-ol (alsoknown as fluconazole, 10.0 g, 32.6 mmol), 1H-tetrazole (6.85 g, 97.8mmol), dibenzyl diisopropyl phosphoramidite (22.55 g, 65.2 mmol) inmethylene chloride (100 ml) was stirred at room temperature under anitrogen atmosphere for 2 hours. The mixture was then cooled to 0° C.,and a solution of 3-chloroperoxybenzoic acid (13.5 g, 50-55% w/w, 39.1mmol) in methylene chloride (50 ml) was added maintaining thetemperature at 0° C. The resulting mixture was allowed to warm to roomtemperature for 1 hour before washing with aqueous sodium metabisulphiteand sodium bicarbonate. After drying (MgSO₄) the solvent was removed andreplaced with methyl isobutyl ketone (37 ml) and tert-butyl methyl ether(74 ml). After granulating at −10° C. for 1 hour the product wasfiltered and washed with ice cold methyl isobutyl ketone and tert-butylmethyl ether (1:3, 15 ml) and dried at 50° C. under vacuum for 18 hoursto give the subtitle compound (16.05 g, 87%), m.p. 93° C.

Found: C, 57.12; H, 4.46; N, 14.85. C₂₇H₂₅F₂N₆O₄P requires C, 57.24; H,4.46; N, 14.84%.

m/z 567 (MH⁺)

¹H NMR (300 MHz, CDCl₃) δ=4.90 (d, 2H), 4.95 (d, 2H), 5.05 (d, 2H), 5.19(d, 2H), 6.58-6.73 (m, 2H), 6.88-6.95 (m, 1H), 7.20-7.30 (m, 4H),7.32-7.38 (m, 6H), 7.80 (s, 2H), 8.36 (s, 2H)

To stirred ethyl acetate (1530 ml) was added2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-ol (alsoknown as fluconazole 306 g, 1.00 mol) and pyridine (237.3 g 3.00 mol)before cooling to 0° C. Phosphorus trichloride (137.4 g, 1.00 mol) wasadded dropwise to the reaction mixture maintaining the temperaturebetween 0-5° C. before allowing the reaction mixture to warm to 15° C.over 30 minutes. Benzyl alcohol (216 g, 2.00 mol) was then added over 30minutes at 15-20° C. After a further 30 minutes hydrogen peroxide (27.5%w/w in water, 373 g) was added maintaining the temperature at 15-20° C.After 30 minutes the aqueous phase was removed and the organic phasewashed with aqueous sodium metabisulphite, dilute hydrochloric acid andwater. The solvent was removed at reduced pressure and replaced withmethyl isobutyl ketone (850 ml) and tert-butyl methyl ether (1132 ml).After granulating at 20° C. for 1 hour and at 0° C. for 1 hour, theproduct was filtered and washed with ice cold tert-butyl methyl ether(2×220 ml) and dried at 50° C. under vacuum for 18 hours to give thesubtitle compound (358g, 63%). The melting point and spectroscopic datawas identical to that stated in method A.

(b) 2-(2,4-Difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)-2-propylDihydrogen Phosphate

A slurry of the compound of step (a) (9.80 g, 17.3 mmol), 5% palladiumon carbon catalyst (50% wet, 1.0 g) and sodium hydroxide (1.38 g, 34.6mmol) in water (26 ml) was hydrogenated at room temperature and 414 kPa(60 p.s.i.) for 20 hours. The solution was filtered through a pad ofcelite (trade mark) and washed with water (5 ml). The toluene wasseparated and the aqueous phase cooled to 0° C. whereupon sulphuric acid(1.70 g, 17.3 mmol) was added. The resulting slurry was granulated at 0°C. for 1 hour and then filtered, washed with water (2×5 ml) and driedunder vacuum at 50° C. to give the title compound (5.80 g, 87%). m.p.223-224° C.

Found: C, 40.28; H, 3.39; N, 21.63. C₁₃H₁₃F₂N₆O₄P requires C, 40.43; H,3.39;N, 21.76%.

¹H NMR (300 MHz, DMSO) δ=5.07 (d, 2H) 5.24 (d, 2H), 6.77-6.83 (m, 1H),7.00-7.18 (m, 2H), 7.75 (s, 2H), 8.53 (s, 2H)

EXAMPLE 2

2-(2,4-Difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)-2-propyl disodiumphosphate

A solution of the compound of Example 1(a) (10.0 g, 17.7 mmol) andsodium acetate (2.90 g, 35.3 mmol) in ethanol (160 ml) and water (20 ml)was hydrogenated over Pearlman's catalyst (1.00 g) at room temperatureand at 345 kPa (50 p.s.i.) for 16 hours. The solution was filteredthrough a pad of celite (trade mark) and the solvents removed at reducedpressure to leave a thick syrup. This was dissolved in ethanol (100 ml)with the aid of sonication and warmed to reflux. The resulting solutionwas allowed to cool slowly and granulate for 1 hour at room temperature.The product was filtered, washed with ethanol (10 ml) and dried undervacuum at 50° C. to give the title compound (4.48 g, 59%).

m.p. 160-162° C.

¹H NMR (300 MHz, D₂O) δ=5.01 (d, 2H), 5.40 (d, 2H), 6.60 (m, 1H), 6.79(m, 1H), 7.11 (m,1H), 7.63 (s, 2H), 8.68 (s, 2H)

EXAMPLE 3

(2R,3S)-2-(2,4-Difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1-(1H-1,2,4-triazol-1-yl)-2-butyldihydrogen phosphate

(a) Dibenzyl(2R,3S)-2-(2,4-difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1-(1H-1,2,4-triazol-1-yl)-2-butylphosphate

A solution of(2R,3S)-2-(2,4-difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol (the compound of Example 7, EP 0440372, also known asvoriconazole, 17.0 g, 48.7 mmol), 4-dimethylaminopyridine (10.2 g, 83.5mmol), 1H-tetrazole (10.2 g, 146 mmol) and dibenzyldiisopropylphosphoramidite (33.6 g, 97.4 mmol) in methylene chloride(100 ml) was stirred at reflux for 2 hours and a further 16 hours atroom temperature under a nitrogen atmosphere. The reaction mixture wasthen washed with hydrochloric acid and then sodium bicarbonate, dried(MgSO₄) and concentrated. The crude product phosphite was purified bycolumn chromatography (silica gel, 300 g, eluting with 3:1 to 1:1hexane:ethyl acetate gradient) to give a pale yellow oil. This wasdissolved in methylene chloride (100 ml) and cooled to −10° C.,whereupon a solution of 3-chloroperoxybenzoic acid (14.8 g, 57% w/w,46.9 mmol) in methylene chloride (100 ml) was added maintaining thetemperature below 0° C. The resulting mixture was allowed to warm toroom temperature for 10 minutes before washing with aqueous sodiummetabisulphite and sodium bicarbonate. After drying (MgSO₄) andconcentrating the crude product was purified by column chromatography(silica gel, 300 g, eluting with ethyl acetate) to give the subtitlecompound as a viscous syrup (17.86 g, 60%).

m/z 610 (MH⁺)

¹H NMR (300 MHz, CDCl₃) δ=1.39 (d, 3H), 4.41 (q, 1H), 4.79 (d, 2H), 4.96(d, 2H), 5.34 (d, 1H), 5.40 (d, 1H), 6.59-6.66 (m, 1H), 6.72-6.82 (m,1H), 7.02-7.18 (m, 3H), 7.23-7.37 (m, 8H), 7.79 (s, 1H), 8.46 (d, 1H),8.52 (s, 1H), 8.90 (d, 1H)

(b)(2R,3S)-2-(2,4-Difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1-(1H-1,2,4-triazol-1-yl)-2-butyldihydrogen phosphate

A solution of the compound of step (a) (5.0 g, 8.83 mmol) in methanol(100 ml) was hydrogenated over Pearlman's catalyst (1.0 g) at roomtemperature and at 414 kPa (60 p.s.i.) for 16 hours. The solution wasfiltered through a pad of celite (trade mark) and concentrated. Thecrude product was redissolved in hot methanol (20 ml) and granulated at0° C. for 1 hour. After filtering and washing with methanol (5 ml) theproduct was dried under vacuum at 50° C. to give the title compound(1.72 g, 49%). m.p. 145-146° C.

¹H NMR (300 MHz, DMSO) δ=1.31 (d, 3H), 4.01 (q, 1H), 5.31 (d, 1H), 5.42(d, 1H), 6.90-6.97 (m, 1H), 7.04-7.14 (m, 1H), 7.20-7.30 (m, 1H), 7.95(s, 1H), 8.70 (d, 1H), 8.73 (s, 1H), 8.89 (d, 1H)

EXAMPLE 4

(2R,3S)-2-(2,4-Difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1-(1H-1,2,4-triazol-1-yl)-2-butyldihydrogen phosphate (Alternative preparation)

(a) Bis(2-chloro-6-fluorobenzyl)(2R,3S)-2-(2,4-difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1-(1H-1,2,4-triazol-1-yl)-2-butylphosphate

A solution of(2R,3S)-2-(2,4-difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol (the compound of Example 7, EP 0440372, also known asvoriconazole, 10.0 g, 28.6 mmol) and 1-methylimidazole (9.40 g, 114mmol) in methylene chloride (30 ml) was cooled to 0° C. whereupon asolution of phosphorus trichloride (4.73 g, 34.4 mmol) in methylenechloride (20 ml) was added, maintaining the temperature below 10° C.After 15 minutes a solution of 2-chloro-6-fluorobenzyl alcohol (12.0 g,74.4 mmol) in methylene chloride (40 ml) was added at between 0-10° C.After 30 minutes hydrogen peroxide (25 ml, 30% solution in water) wasadded dropwise maintaining the temperature below 20° C. with cooling.After a further 1 hour the reaction mixture was separated and theorganic phase was washed with water (2×100 ml), dried (MgSO₄) andconcentrated. The resulting viscous oil was granulated with tert-butylmethyl ether (60 ml) for 2 hours at 0° C. The product was filtered,washed with tert-butyl methyl ether (20 ml) and dried at 50° C. in vacuofor 18 hours to give the subtitle compound as a white crystalline solid(18.1 g, 88% yield), m.p. 140-141° C.

¹H NMR (300 MHz, CDCl₃) δ=1.39 (d, 3H), 4.33 (q, 1H), 5.08 (d, 1H), 5.13(d, 1H), 5.27(d, 1H), 5.31 (d, 1H), 5.32 (d, 1H), 5.42 (d, 1H),6.60-6.75 (m, 2H), 6.92-7.07 (m, 2H), 7.11-7.37 (m, 5H), 7.81 (s, 1H),8.44 (d, 1H), 8.61 (s, 1H), 8.91 (s, 1H)

(b)(2R,3S)-2-(2,4-Difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1-(1H-1,2,4-triazol-1-yl)-2-butyldihydrogen phosphate

A mixture of the compound of step (a) (50 g, 70 mmol), sodium hydroxide(8.40 g, 210 mmol) and 5% palladium on carbon catalyst (10 g) in toluene(450 ml) and water (150 ml) was hydrogenated at room temperature and 414kPa (60 p.s.i.) for 24 hours. The reaction mixture was filtered throughcelite (trade mark) and the toluene layer separated and discarded. Theaqueous layer was then washed with methylene chloride (2×75 ml) andtoluene (2×75 ml) and then cooled to 0° C. whereupon sulphuric acid(10.3 g, 105 mmol) was added. After granulating at 0° C. for 1 hour theproduct was filtered, washed with water (60 ml) and dried under vacuumat 50° C. for 16 hours to give the title compound (20.5 g, 68%). Theproton NMR data was identical to that obtained in Example 3(b).

Found: C, 44.48; H, 3.45; N, 16.19. C₁₆H₁₅F₃N₅O₄P requires C, 44.77; H,3.52;N, 16.31%.

EXAMPLE 5

(2R,3S)-2-(2,4-Difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1-(1H-1,2,4-triazol-1-yl)-2-butyl(2-hydroxyethyl)trimethylammonium Hydrogen Phosphate Dihydrate

To a stirred slurry of the compound of Example 3(b) (214.7 g, 500 mmol)in acetone (2070 ml) was added a solution of choline bicarbonate (75%w/w in water, 110 g, 500 mmol) over 10 minutes. The slurry was warmed toreflux for 20 minutes, filtered through a pad of celite (trade mark) toremove any insoluble material then cooled to 20° C. and granulated for 1hour. The resulting product was collected by filtration, washed withacetone (2×250 ml) and dried at 20° C. under vacuum for 18 hours to givethe title compound (233.3 g, 74%) m.p. 114-115° C.

¹H NMR (300 MHz, DMSO) δ=1.23 (d, 3H), 3.07 (s, 9H), 3.38 (t, 2H), 3.60(q, 1H), 3.78 (q, 2H), 5.50 (s, 2H), 6.72-6.80 (m, 1H), 6.94-7.02 (m,1H), 7.36-7.42 (m, 1H), 7.82 (s, 1H), 8.59 (d, 1H), 8.78 (d, 1H), 9.35(s, 1H)

EXAMPLE 6

2-(2,4-Difluorophenyl)-1-{3-[(E)-4-(2,2,3,3-tetrafluoropropoxy)styryl]-1H-1,2,4-triazol-1-yl}-3-(1H-1,2,4-triazol-1-yl)-2-propyldihydrogen phosphate

(a) Dibenzyl2-(2,4-difluorophenyl)-1-{3-[(E)-4-(2,2,3,3-tetrafluoropropoxy)styryl]-1H-1,2,4-triazol-1-yl}-3-(1H-1,2,4-triazol-1-yl)-2-propylphosphate

2-(2,4-Difluorophenyl)-1-{3-[(E)-4-(2,2,3,3-tetrafluoropropoxy)styryl]-1H-1,2,4-triazol-1-yl}-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (the racemate of Example 19, EP 0472392, 475 mg, 0.88 mmol),1H-tetrazole (185 mg, 2.64 mmol) and dibenzyl diisopropylphosphoramidite(607 mg, 1.76 mmol) in methylene chloride (5 ml) was stirred at roomtemperature under a nitrogen atmosphere for 20 hours. The mixture wasthen cooled to 0° C., and hydrogen peroxide (1.0 ml, 30% solution inwater) was added dropwise maintaining the temperature below 20° C. Theresulting mixture was stirred at 20° C. for 30 minutes before separatingthe organic layer, which was washed with water, dried (MgSO₄) and thesolvent evaporated. The resulting pale yellow oil was purified by columnchromatography (silica gel, eluting with ethyl acetate/hexane) to givethe subtitle compound as a viscous syrup (595 mg, 84%).

¹H NMR (300 MHz, CDCl₃) δ=4.37 (t, 2H), 4.91 (d, 2H), 4.97 (d, 2H), 5.02(d, 1H), 5.07 (d, 1H), 5.16 (d, 1H), 5.18 (d, 1H), 6.05 (tt, 1H),6.59-6.78 (m, 2H), 6.82 (d, 1H), 6.90 (d, 2H), 6.91-7.00 (m, 1H),7.21-7.38 (m, 10H), 7.42 (d, 2H), 7.42 (d, 1H), 7.79 (s, 1H), 8.28 (s,1H), 8.39 (s, 1H)

(b)2-(2,4-Difluorophenyl)-1-{3-[(E)-4-(2,2,3,3-tetrafluoropropoxy)styryl]-1H-1,2,4-triazol-1-yl}-3-(1H-1,2,4-triazol-1-yl)-2-propyldihydrogen phosphate

A solution of the compound of step (a) (298 mg, 0.37 mmol) in methylenechloride (5 ml) was cooled to 0° C. and then treated withbromotrimethylsilane (254 mg, 1.66 mmol) and pyridine (180 mg, 3.10mmol). The resulting mixture was stirred at 0° C. for 3 hours and thenquenched with water (1 ml) containing sodium hydroxide (96 mg, 2.41mmol). The mixture was then acidified with dilute sulphuric acid and theproduct extracted into ethyl acetate. After washing with brine, theethyl acetate phase was dried (MgSO₄) and the solvent evaporated to givethe title compound as a pale yellow foam (202 mg, 88%).

EXAMPLE 7

(2RS,3RS)-3-(4-[4-Cyanophenyl]thiazol-2-yl)-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)-2-butyldihydrogen phosphate

(a) Dibenzyl(2RS,3RS)-3-(4-[4-cyanophenyl]thiazol-2-yl)-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)-2-butylphosphate

3-[4-(4-Cyanophenyl)thiazol-2-yl]-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol(Example 88, EP 0667346, 900 mg, 2.06 mmol), 1H-tetrazole (432 mg, 6.18mmol), 4-dimethylaminopyridine (100 mg, 0.82 mmol) and dibenzyldiisopropylphosphoramidite (1.42 g, 4.12 mmol) in methylene chloride (10ml) were refluxed under a nitrogen atmosphere for 20 hours. The mixturewas then cooled to 0° C., and hydrogen peroxide (2.5 ml, 30% solution inwater) was added dropwise maintaining the temperature below 20° C. Theresulting mixture was stirred at 20° C. for 30 minutes before separatingthe organic layer, which was washed with water, dried (MgSO₄) and thesolvent evaporated. The resulting pale yellow oil was purified by columnchromatography (silica gel, eluting with ethyl acetate/hexane) to givethe subtitle compound as a viscous syrup (732 mg, 51%).

¹H NMR (300 MHz, CDCl₃) δ=1.40 (d, 3H), 4.38 (q, 1H), 4.81-4.96 (m, 4H),5.40 (d, 1H), 5.43 (d, 1H), 6.62-6.71 (m, 1H), 6.74-6.82 (m, 1H),7.15-7.37 (m, 10H), 7.58 (s, 1H), 7.62 (d, 2H), 7.73 (s, 1H), 7.97 (d,2H), 8.48 (s, 1H)

(b)(2RS,3RS)-3-(4-[4-Cyanophenyl]thiazol-2-yl)-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)-2-butyldihydrogen phosphate

A solution of the compound of step (a) (310 mg, 0.44 mmol) in methylenechloride (5 ml) was cooled to 0° C. and then treated withbromotrimethylsilane (303 mg, 1.98 mmol) and pyridine (215 mg, 3.70mmol). The resulting mixture was stirred at 0° C. for 3 hours and thenquenched with water (1 ml) containing sodium hydroxide (115 mg, 2.87mmol). A yellow precipitate was formed, which was isolated by filtrationand then partitioned between dilute sulphuric acid and methylenechloride. The organic phase was washed with brine, dried (MgSO₄) and thesolvent evaporated to give the title compound as a pale yellow solid (80mg, 35%).

¹H NMR (300 MHz, DMSO) δ=1.38 (d, 3H), 4.22 (q, 1H), 5.37 (d, 1H), 5.41(d, 1H), 6.88-6.97 (m, 1H), 7.09-7.19 (m, 1H), 7.31-7.40 (m, 1H), 7.80(s, 1H), 7.87 (d, 2H), 8.05 (d, 2H), 8.32 (s, 1H), 8.65 (s, 1H)

EXAMPLE 8

(2R,3S)-2-(2,4-Difluorophenyl)-3-[4-(1-methylpyrazol-5-yl)phenyl]-1-(1,2,4-triazol-1-yl)-2-butyldisodium phosphate

a) O,N-dimethyl-4-iodobenzenehydroxamic acid A solution of pyridine (104g, 1.32 mol) in dichloromethane (150 ml) was added dropwise to asuspension of 4-iodobenzoyl chloride (251 g, 0.94 mol) andN,O-dimethylhydroxylamine hydrochloride (97 g, 0.94 mol) indichloromethane (850 ml) at 0° C. The mixture was allowed to warm toroom temperature and was stirred for 18 hours. The solution wasevaporated under reduced pressure, the residue was dissolved in ethylacetate (11), and was then washed with dilute hydrochloric acid (2N,3×400 ml) and saturated sodium bicarbonate solution (300 ml) and dried(Na₂SO₄). The organic extract was evaporated under reduced pressure. Theresidue was purified by distillation to yield the subtitle compound (241g, 93%) as a yellow oil, b.p. 130° C. (0.1 mm Hg), which wascharacterized by ¹H NMR.

(b) 2-(2,4-Difluorophenyl)-1-(4-iodophenyl)ethanone

2,4-Difluorobenzyl bromide (23.7 ml, 0.114 mol) was added dropwise to astirred mixture of magnesium turnings (8.1 g, 0.183 mol) in dry ether(300 ml) under nitrogen. The mixture was warmed initially until reactionstarted, and thereafter the bromide was added at such a rate as tomaintain a gentle reflux. After 1 hour, the resulting solution of theGrignard reagent was added dropwise at −78° C. to a solution ofO,N-dimethyl-4-iodobenzenehydroxamic acid [see step (a)] (45.7 g, 0.157mol) in dry ether (300 ml), and the mixture was allowed to warm slowlyto room temperature overnight. The mixture was partitioned betweensaturated aqueous ammonium chloride and ethyl acetate, and the organicsolution was separated, dried (MgSO₄) and concentrated under reducedpressure, to give the title compound as a white solid, 38.71 g (69%),which was characterised by ¹H-N.M.R. spectroscopy.

(c) 2-(2,4-Difluorophenyl)-1-(4-iodophenyl)prop-2-enone

Bis(dimethylamino)methane (8.78 ml, 0.075 mol) was added dropwise to astirred suspension of 2-(2,4-difluorophenyl)-1-(4-iodophenyl)ethanone[17.73 g, 0.04595 mol, from step (b)] in acetic anhydride (23.1 ml,0.248 mol) at room temperature. There was an exothermic reaction, andthe temperature of the mixture rose to 60° C. After the end of theaddition, the mixture was stirred at room temperature for 35 minutes,and then iced water was added to hydrolyse the excess acetic anhydride.After a further 30 minutes, the product was extracted into ethylacetate, and the extracts were washed with dilute hydrochloric acid,saturated aqueous sodium bicarbonate, dried (MgSO₄), and concentratedunder reduced pressure, to give the title compound as a white solid(17.03 g, 93%), which was characterised by ¹H-N.M.R. spectroscopy.

(d) 2-(2,4-Difluorophenyl)-2-(4-iodobenzoyl)oxirane

Benzyltrimethylammonium hydroxide (3.44 ml, 40% aqueous solution, 8.2mmol) was added in one portion to a solution of2-(2,4-difluorophenyl)-1-(4-iodophenyl)prop-2-enone [37.3 g, 100.8 mmol,from step (c)] and t-butylhydroperoxide (36.6 ml, 3M intrimethylpentane, 109 mmol) in toluene (550 ml) at room temperature.After 2 hours, the mixture was washed with water (2×500 ml), dried(MgSO₄) and concentrated under reduced pressure to give the titlecompound as a white solid (37.46 g, 96%), which was characterised by¹H-N.M.R. spectroscopy.

(e) (2,4-Difluorophenyl)-2-[1-(4-iodophenyl)ethenyl]oxirane

n-Butyllithium (50 ml, 2.5 M in hexane, 125 mmol) was added dropwiseover 10 minutes to a stirred suspension of methyltriphenylphosphoniumbromide (45.0 g, 126 mmol) in dry THF (600 ml) under nitrogen at −70° C.The mixture was allowed to warm to −20° C., over 20 minutes, then asolution of 2-(2,4-difluorophenyl)-2-(4-iodobenzoyl)oxirane [37.46 g, 97mmol, from step (d)] in dry THF (200 ml) was added over 5 minutes. Themixture was allowed to warm to room temperature and stirred for 84hours. 10% Aqueous ammonium chloride (500 ml) was added, and the mixturewas concentrated under reduced pressure. The product was extracted intoethyl acetate and the combined extracts were dried (MgSO4) andconcentrated under reduced pressure. The solid residue was treated withboiling hexane (3×500 ml), and the residual solid discarded. The hexanesolutions were combined, filtered through a short pad of silica gel, andconcentrated under reduced pressure to give the title compound as ayellow oil (34.3 g, 92%), which was characterised by ¹H-N.M.R.spectroscopy.

(f)2-(2,4-Difluorophenyl)-3-(4-iodophenyl)-1-(1H-1,2,4-triazol-1-yl)-3-buten-2-ol

Sodium 1,2,4-triazole (12.15 g, 133 mmol) was added to a solution of(2,4-difluorophenyl)-2-[1-(4-iodophenyl)ethenyl]oxirane [34.3 g, 89mmol, from step (e)] in dry DMF (350 ml) under nitrogen at 70° C. Themixture was stirred for 5 hours, cooled, and the solvent removed underreduced pressure. The residue was partitioned between ether (800 ml) andwater (2×500 ml). The organic solution was dried (MgSO₄), filtered, andsilica gel (60-200μ, 75 g) was added. The ether was removed underreduced pressure and the residual solid was applied to the top of asilica gel column (40-60%, 300 g) and the product was eluted usinghexane and increasing amounts of ethyl acetate (0-75%). The product wasobtained as a white foam (23.8 g, 61%), which was characterised by¹H-N.M.R. spectroscopy.

(g)(R)-2-(2,4-Difluorophenyl)-3-(4-iodophenyl)-1-(1H-1,2,4-triazol-1-yl)-3-buten-2-ol(+)-3-bromocamphor-10-sulphonate

A solution of (+)-3-bromocamphor-10-sulphonic acid (36.3 g, 0.110 moles)in IMS (40 ml) was added to a solution of the product of step (f) (50 g,0.110 moles) in IMS (300 ml). After seeding, the resulting slurry wasgranulated for 20 hours at room temperature. A white solid (22 g, 0.03moles) was collected by filtration after further granulating for 1 hourat low temperature. The chiral purity was assessed as 95% ee by chiralHPLC using a Chiralcel™ OD column and eluting with ethanol/hexane[40:60].

(h)(R)-2-(2,4-Difluorophenyl)-3-(4-iodophenyl)-1-(1H-1,2,4-triazol-1-yl)-3-buten-2-ol

The product of step (g) (206.5 g, 0.27 moles) was added to methylenechloride (620 ml) and basified with 40% NaOH. The mixture was stirredfor 15 minutes at room temperature and separated. The aqueous phase wasre-extracted with methylene chloride (310 ml). The organic productsolution was washed with water (620 ml) and concentrated to a volume of245 ml. To the stirred and seeded concentrate at room temperature wasadded hexane (2450 ml) at a steady rate. The resulting slurry wasgranulated at 5° C. for 1 hour. Filtration afforded a white solid (117.4g, 0.26 moles) which was characterized by ¹H NMR spectroscopy.

(i)(2R)-2-(2,4-Difluorophenyl)-3-[4-(1-methylpyrazol-5-yl)phenyl]-1-(1,2,4-triazol-1-yl)-3-buten-2-ol

nBuLi (1.6N, 24.1 ml, 0.04 moles) was added to a solution of1-methylpyrazole (3.28 g, 0.04 moles) in THF (370 ml) at −70° C. keepingthe temperature below −60° C. and stirred for 30 minutes. Maintaining atemperature below −40° C., a solution of zinc chloride (0.5N, 77.1 ml,0.04 moles) was added, followed by palladiumtetrakis(triphenylphosphine) (15% w/w, 0.9 g). Still keeping thetemperature below −40° C., a solution of the product of step (h) (6 g,0.013 moles) in THF (36 ml) was added at a steady rate. The reaction wasallowed to warm to room temperature and then refluxed for 2 hours. Aftercooling to room temperature, the reaction was quenched with acetic acid(12 ml) and water (120 ml) keeping the temperature below 25° C. Thereaction mixture was evaporated under reduced pressure to remove theTHF. The product was extracted with methylene chloride (120 ml) and theaqueous phase further extracted with methylene chloride (50 ml). Thecombined organic extracts were washed with water (2×120 ml) andconcentrated to give an oil. To a stirred filtered solution of the oilin ethyl acetate (100 ml) was added 5-sulphosalicylic acid (3.3 g, 0.13moles) in IPA (10 ml). The resulting mixture was stirred at roomtemperature for ½ hour. The resulting filtered solid was repulped inethyl acetate (50 ml) and recrystallized from IPA (60 ml) to afford awhite solid (7.2 g, 0.01 moles). The solid was added to methylenechloride (35 ml) and water (50 ml) and basified with 40% NaOH. Themixture was stirred at room temperature for 15 minutes and separated.The aqueous phase was re-extracted with methylene chloride (25 ml) andthe combined organic extracts washed with water (35 ml). The organicproduct solution was concentrated to a foam and characterized.

[α]_(D)=−25.6°

C₂₂H₁₉F₂N₅O.0.5H2O requires C, 63.45; H, 4.84; N, 16.82. Found C, 63.92;H, 4.86; N, 16.64.

(j)(2R,3S)-2-(2,4-Difluorophenyl)-3-[4-(1-methylpyrazol-5-yl)phenyl]-1-(1,2,4-triazol-1-yl)butan-2-ol

A solution of the product of step (i) (2.0 g, 5 mmol) in ethanol (50 ml)was hydrogenated at 50 psi (333 KPa) pressure over 5% palladium oncharcoal (0.2 g) for 18 hours at 50° C. A further batch of catalyst (0.2g) was added, and the hydrogenation was continued for a further 18hours. The mixture was filtered through Arbocel™ and the filtrateevaporated under reduced pressure. The residue was chromatographed onsilica by gradient elution with ethyl acetate/hexane/diethylamine(0:95:5→65:33:2). Fractions containing the desired product were combinedand evaporated under reduced pressure. The residue was dissolved andre-evaporated from ethyl acetate (×3) then from ether (×3) to yield acolourless solid. The solid was recrystallised from aqueous ethanol togive the subtitle compound (1.25 g, 62%) as a colourless solid, m.p.144-145° C., [α]_(D)=−107° (c=0.1%, CH₂Cl₂, 25° C.).

Elemental analysis (%)

Found:C, 64.26; H, 5.13; N, 17.07

C₂₂H₂₁F₂N₅O requires: C, 64.54; H, 5.17; N, 17.10

[This compound is also disclosed as Example 67 in co-pendingInternational Patent Application PCT/EP96/02470].

(k) Dibenzyl(2R,3S)-2-(2,4-difluorophenyl)-3-[4-(1-methylpyrazol-5-yl)phenyl]-1-(1,2,4-triazol-1-yl)-2-butylphosphate

A solution of the product of step (j) (2.0 g, 4.4 mmol), dibenzyldiisopropylphosphoramidite (2.28 g, 6.6 mmol), tetrazole (0.92 g, 13.2mmol) and 4-dimethylaminopyridine (50 mg) in dichloromethane (30 ml) washeated under reflux for 13 hours. The reaction mixture was cooled (0°C.) and m-chloroperbenzoic acid (1.52 g, 8.8 mmol) added. The solutionwas stirred at 0° C. for a further hour then allowed to warm to roomtemperature. The reaction mixture was washed with aqueous sodiumsulphite solution (10%, 30 ml), saturated sodium bicarbonate solution(30 ml) and brine (30 ml). The organic layer was dried (Na₂SO₄) andsolvent was evaporated in vacuo to give an oil. Purification by columnchromatography (silica gel, 45 g, eluting with toluene to 3.5%diethylamine in toluene gradient) gave the required product as acolourless oil (0.8 g, 27%), m/z 671 (M⁺+1). ¹H N.M.R. (CDCl₃) δ=1.3 (d,3H); 3.8 (s, 3H); 3.85 (q, 1H); 4.8 (m, 2H); 4.9 (m, 2H), 5.2 (s, 2H);6.25 (s, 1H); 6.6 (m, 1H); 6.8 (m, 1H); 7.05 (m, 1H); 7.15 (m, 2H);7.2-7.35 (m, 12H); 7.5 (s,1H); 7.8 (s,1H); 8.23 (s,1H).

(l) (2R,3S)-2-(2,4-Difluorophenyl)-3-[4-(1-methylpyrazol-5-yl)phenyl]-1-(1,2,4-triazol-1-yl)-2-butyldisodium phosphate

A suspension of the product of step (k) (0.5 g, 0.75 mmol) and sodiumacetate (0.14 g, 1.65 mmol) in ethanol (20 ml) was hydrogenated over 5%Palladium on carbon (75 mg) at room temperature and 333 KPa (50 p.s.i.)for 24 hours. Tlc analysis showed incomplete reaction and the catalystwas filtered off through a pad of filter aid (Arbacel, trade mark).Pearlman's catalyst (75 mg) was then added and hydrogenation continuedfor a further 72 hours. The catalyst was filtered off through a pad ofArbacel and solvent removed in vacuo. The residue was dissolved indichloromethane (20 ml) and filtered through a pad of filter aid(Hiflow, trade mark) to remove excess sodium acetate. Solvent wasevaporated in vacuo and after trituation with diethyl ether, the titlecompound obtained as a white solid (0.250 g, 68%). Found: C, 46.66; H,4.87; N, 11.82; C₂₂H₂₂F₂N₅Na₂O₄P. 0.09 Et₂O requires C, 46.62; H, 4.36;N, 12.16%. ¹H N.M.R. (DMSO) δ=1.2 (d, 3H); 3.75 (q, 1H); 3.8 (s, 3H);5.1, 5.5 (AB system, 2H); 6.3 (s, 1H); 6.6 (m, 1H); 6.9 (m, 1H); 7.2,7.4 (AB system, 4H); 7.4 (m, 3H); 7.45 (m,1H); 7.6 (s,1H); 9.1 (s,1H).

EXAMPLE 9

The aqueous solubilities of the compounds of Examples 1, 3 and 8 (in theform or their disodium salts) were compared with the solubilities oftheir respective parent (non-phosphorylated) compounds (in free baseform). The results are shown in the following table.

Compound Solubility (mg/ml) Example 1 >150 Parent 2 Example 3 >150Parent 0.6 Example 8 >50 Parent 0.01

EXAMPLE 10

Aqueous Formulation for i.v. Injection

Ingredient mg/ml Compound of Example 1 100.00 Sodium hydroxide 22.80Hydrochloric acid, concentrated q.s.* Water for injections to 1.00 ml*The pH range of the resulting solution is adjusted to between pH 8.5and 9.5 by the addition of a sufficient quantity of 5 M hydrochloricacid.

What is claimed is:
 1. A compound, which is:(2R,3S)-2-(2,4-difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1-(1H-1,2,4-triazol-1-yl)-2-butyldihydrogen phosphate; or a pharmaceutically acceptable salt thereof. 2.A compound which is: Alkali metal salt of(2R,3S)-2-(2,4-difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1-(1H-1,2,4-triazol-1-yl)-2-butyldihydrogen phosphate.
 3. A compound which is:(2R,3S)-2-(2,4-difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1-(1H-1,2,4-triazol-1-yl)-2-butyldisodium phosphate.